Enzymic substance and process of making the same



UNITED STATES PATENT OFFICE.

JOKICHI TAK AMINE, OF NEW YORK,

Specification of Letters Patent.

N. Y., AND .TOKICHI TAKAMINE, JR., OF CLIFTON, NEW JERSEY.

ENZYMIG SUBSTANCE AND PROCESS OF MAKING THE SAME.

1,391,219. No Drawing. Application filed January 23, 1918. Serial No.213,431.

To all whom it may concern:

Be it known that and JOKICHI TAKAMINE, the Emperor of Japan, 5spectively. in the city,

New York, and Clifton, State of New Jersey,

JoKIoHI TAKAMINE Jr., subjectsof and residing, recounty, and State ofcounty of Passaic, have made a certain new and useful invention iEnzymicsubstance and Processes of Making the Same, of which thefollowing is a specification.

This invention relates to enzymic sub-- stances, and the process ofmaking the same.

The object of the invention is to produce an enzymic substance suitablefor use in various lines of industry where the enzymic property of thesubstance is to be utilized.

A further object produce an enzymie industrial arts, whi

of the invention is to s I bstance for use in the ch is stable and can bpreserved without deterioration.

A further object i s to produce an enzymic substance of the naturereferred to from a fungus growth. Other objects,

of theinvention will appear actertistics hereinafter more fully setforth and finally pointed out in the appended claims.

In carrying out spores of suitable our invention the seed microscopicfungi are sown on suitable culture media and propagated under suitableconditions of temperature, humidity and handling and for a suitableperiod of time, for the fungus to at- H -which it develops its tain thatstage in greatest enzymic po stances contained in wer. I The enzymicsubthe fungus are then extracted by suitable solvents and by suitabletreatment so as to obtain an enzymic product for use in the industrialarts, as we will more fully explain hereinafter. The seed spores of thefungi genera Aspergillus, ticularly tium 'org zae, or our invention.

Pe'mwm'llium or M ucor, and, parthe seed spores of the species E uroaresuitable for the purposes Comminuted or broken grains of the cereals,such as wheat, corn, oats, barley,

and preferably such broken or. comminuted grains from which the greaterpart of the.

The mass or culture medium to be employed is first suitably moistenedwith water and thoroughly sterilized. This may be accomplished withsteam. The seed spores of the desired fungus are sown upon thesterilized mass while the latter is in a moistened state, preferablycontaining from thirty to sixty per cent. of moisture. The sterilizingof the mass isnot essential and may be omitted although a more abundantand healthier growth of fungus is secured Patented Sept. 20, 1921.

and a greater development of enzymic power attained by first sterilizingthe culture medium. In the sterilizing operation bacteria contained inthe culture medium, and which tend to cause undesirable, fermenting ordecomposing action in when produced by the subsequent treatment of themass, are more or less destroyed or rendered inert.

After sowing the fungus seed spores upon the culture medium the entiremass is bedded up to a thickness of from six to twentyfour inches. Inthe case of wheat bran, shorts, middlings, &c., the bedding up of themass is not essential. The mass is maintained in amoist atmosphere andat a uniform temperature not to exceed 45 C. There are different methodsof carrying on thepropagation and development of the fungus growth. Oneis the still growth method. Anotheris growing in motion. In the case ofstill growth the mass 's spread to a thickness of from one to two inchesand the fungus allowed to develop and grow. In the motion growing methodthe mass which may vary in thickness up to two or three feet, isconstantly maintained in motion by stirring or otherwise in such mannerasto cause every portion of the mass to be exposed tothe air and to thecooling effect of the air, thereby. preventing the enzymic substancethemass from developing too great a heat.

In the course offrom eight to twenty hours the spores begin to sproutand within twenty to thirty hours the growth of fungus becomes abundant.Carbonic acid gas and heat are evolved during this period. Within thirtto sixt hours enzymic pro rties are eveloped 1n the culture medium y thegrowth of the fungus. The enzymes produced are mostly soluble in waterand possess various characteristics such as diastatic, proteol tic,fat-s litting, milk-coagulating, and t e like. he relative proportionsof these characteristics or properties vary with different species ofspores employed, and also, to more or less extent, ac-

cording to the temperature, humidity, han dling, and other conditionsobserved during the propagation and growth of the fungus.

The various enzymes are separated. from the mass. Being soluble in waterthis separation of the enzymes may be effected by lixiviating the masswith water. The solution thus obtained may be concentrated in anyconvenient manner. For example, a desirable degree of concentration maybe effected by percolating the same aqueous solution through successivebatches or masses of the culture medium upon which the fungus has beengrown.

Of the enz mes contained in the solution thus obtaine diastatic enzymeusually predominates, followed by. proteolytic, milkcoagulating,fat-splittin and others, cited in their relative streng i. I a

The solution or extract obtained as described is suitable for use inmany indus-' trial arts, for example, for destarching woven cottonfabrics, where utilization is made of its property of rendering starchsoluble, that 1s, its diastatic property; de-

' gumming silk fibers and woven goods, where fermentation, whichactually destroys t utilization is made of its erty; in bread making,where utilization is made of its powers of liquefying and saccharifyingstarch; and for numerous other proteolytic propesired. This solution,however, is more or less unstable, that is, in a comparatively limitedperiod of time decomposition sets up therein, resulting in various kindsof greatly impairs, if not e valuable enzymic properties of thesolution. This is a serious ob'ection since it necessitates the use ofthe .so ution as soon as it is made and conseimposes, a serioussubstance.

Eyen afterevaporation of quently a fresh quantity must be made up eachtime it is re ui red for use, and this rawbaok upon thepracticalavailability of "the enzymic solution or the solution, to asyrupy or os1t 1on and conseunder a vacuum ofotherwise, semi-solidstate, gdecom 66' quent fermentation stil takes place, andfermentationand without im 1 I I moreover, during evaporation a large percentage ofenzymic power is lost either bydecomposition orby the heat appliedduring evaporation. I

It is. among the. special purposes of our invention to produce anenzymic substance of the nature referred to which is stable and can bepreserved without decomposition and mic power thereof.

enz

e have discovered that decomposition pairing the I a I and resultingfermentation'in the enz mic 1 our invention. This elimination may beeffected in-various ways. One eflicacious' and convenient way is tosecure a concentrated solution by employing the extract obtained fromone portion of culture medium to effect extraction from successiveportions of such medium until the extract contains from fifteen tothirty per cent. of solid matter, where wheat bran, shorts, middlings,or combinations of such materials are employed for the culture medium Toa given quantity of the enzymic solution an inert silicious materialsuch as infusorial earth, fullers earth, or other similar material inthe proportion of one-tenth of one The in usorial earth, or othersimilar material employed, should be first thoroughly sterilized. hemassis thoroughly stirred or agitated and is then filtered by suitablemeans. The filtering operation is repeated as often as may be requireduntil a perfectly clear transparent solution is obtained. Theelimination of the spores and bacteria thus accomplished leaves asolution possessing the desired enzymic power and which is suitable foruse and in which decomposition and fermentation will not occur rapidly,and. -wh1ch, therefore, siderable period can be preserved for a contion.It some times occurs that even after repeated filtering some deleteriousbacteria still remain in the solution. To secure a still morestablesol-ution which can be preserved without deterioration it isnecessary to sterilize or render all bacteria contained therein inert.The difficulty in accomplishing this resides in the danger of impairingthe enzymic power of the substance. sterilizing antls'eptics such asformaldehyde, sulfurous acid, and other suitable bactericidesmay be usedfor the purpose, but the danger in the use of such antiseptics is thatif a sufficient quantity of antiseptic isused to'thoroughly efl'ectsterilization of the undesirable bacteria' contained in the enzymicsolution the enzymic power of the solution is materially of time withoutdeterioraer cent. to two per cent. is added.

, tiseptic in suflicient quantity, or a temperaplication of a properamount After passing throu' h a desired length of the coil, say onehundred feet, the solution elude outer contamination.

' is a clear transpargent dark amber 4 somewhat nutty taste,

ture of sufiiciently high degree, to insure elimination of the injuriouseffects of undesired bacteria present in the solution, also i greatlyreduces the value of the solution by diminishing its enz mic power ifnot actually destroying suc 'ower. While, therefore, an antiseptic, sucsulfurous acid in the proportion of a small fraction of one percent.,-added to the clear transparent solutionaids in increasing thestability of the solution, we have discovered, that by a novel andproper combination of antiseptic in suitable quantity, and the a ofheat, t e joint effect of the two agencies, when properly applied underproper conditions, we are able to secure the best results in obtainingthe desired efi'ect of stability of the enzymic substances, withoutinjury to the enzymic power thereof. In practice we have found that thebest results are attained when a small percentage, say one part ofantiseptic, sulfurous acid, for example, in from one to ten thousandparts of the filtered transparent clear enzymic substance is added tothe latter and thoroughly mixed therein, and the mixture is then heatedto say 45 C. We

have found that this relative quantity of antiseptic alone, nor thisdegree of heat alone, is not suflicient to kill the bacteria, nor tosubstantially impair the enzymic power of but the combination theenzymic substance, of the two conditions of heat and antiseptic referredto, is sufiicient to render the enzymic substancestable and capable ofbeing preserved for future use in perfect condition and withoutimpairment of its enzymic power. We prefer to preserve the enzymicsubstance obtained and treated as above described,- free fromcontaminating or decomposing influences by properly sterilizing andsealing the containers in which it "is preserved.

In practice, the enzymic. form of a solution, into which-the desiredamount of antiseptic ismixed, is passed through a coil, say of copper,which is kept in water heated to the required temperature.

into a sterilized container, full, is properly sealed to exis deliveredwhich, when The product obtained colored liquid having a with a sli htlymusty odor, the color, taste and 0 or varying as formaldehyde, or

rial, suitably substance, in the somewhat according to the degree of conble starch, and then into various forms of dextrins, and eventually intosugars. The different stages of these conversions may be renderedvisible by the addition of a weak solution of iodin to the mixture ofstarch and the enzymic solution. The self gives an insoluble blue colorwhile the stage'of soluble starch is indicated by a soluble bluecolorwhich changes into various stages of purple to brown as the converting80131011 progresses, the color eventually disappearing when the stage ofsugars is reached in the conversion. The enzymic solution when obtainedom a culture medium of wheat, bran, or other similar matesteriliz'ed,gives vno coloration when an alcoholic solution ofgum guayacol andhydrogen peroxid is added thereto. This is a characteristic whichdistinguishes this enzymic solution from a solution ob- .tained frommalt or germinated grain which would, in like case, give a blue color.Phosphate constitutes alarge percentage of the mineral content of theenzymic solution, v

starch it-' whereas an enzymic solution obtained from malt contains acomparatively small quan-' t1ty 0f phosphate. As compared with theordinary malt extract of commerce the enzymic solution of our inventioncontains a small amount of sugar and a comparatively large amount of'proteid orcomparatively anic matter.

The proteolytic properties of the enzymic substance of our invention areshown by the digesting action it exerts upon chopped meat. ithin a shortperiod of time after the enzymic substance is added to the meat, saywithin about five minutes, digestion of the meat starts in, peptonesbeing formed at first followed by amino acids, &c.

The milk coagulating property of the substance is easily demonstrated bythe addition of a small amount of the substance, say from one to two percent., to themilk. he milk coagulates to whey and eventually into,cheese, a good cheese forming in about one invention, and desire tosecure by Letters Patent, is,-

1. The process which consists in propagating a fungus upon aculturemedium, then extracting the soluble content of the mass, thenadding an inert silicious substance to the extract and filtering thesame toproduce 3. The process which consists in propa gating a fungusupon a culture medium, then extracting the soluble content of theculture medium having the fun us growth thereon, and finally adding. sulurous acid to the extract.

4. The process which consists in propagating a fungus upon a culturemedium,

then extracting the soluble content of the culture medium having thefungus growth thereon, and finally. subjecting the extract to thecombined effect of heat and an antiseptic.

5. The process which consists in propagating a fungus upon a culturemedium, then extracting the 'soluble content of the culture mediumhaving the fungus growth thereon, then mixing sulfurous acid with theextract and heating the mixture.

6. The process which consists in propaating a fungus upon a culturemedium, and

extracting with water the soluble content of the resulting mass, thenfiltering the extract, and mixing sulfurous acid therewith and heatingthe mixture'to a temperature insufficient to impair the enzymic power ofthe solution.

7. The process which consists in propagating a fungus upon a culturemedium, and extracting with water the soluble content of the resultingmass, then filtering the'extract,

and finally mixing with thefiltered solution an antiseptic ininsufficient quantitygand heating the mixture to an insuflicient degreeto impair the enzymic power of the solution.

8. The process which consists inpropagating a fungus upon a culturemedium, then extracting the soluble content of the mass, then filteringthe extract into a trans- Parent clear liquid and finally subjecting theiquid to the combined action of an antiseptic and heat;

9. The process which consists in propagating a fungus upon a culturemedium, then successively extracting the. soluble content of differentportions of the mass'with the same liquid, and finally subjecting theextract to the combined action of an anti- 11. The processwhich-consists of propagating a fungus upon a cuture medium, thenlixiviating said mass with water and concentrating the same, and finallystabilizing the concentrate by subjection to the action of heat and anantiseptic.

12. The process which consists of propa.

gating a fungus upon a culture medium,

then lixiviating said mass with water, and

concentrating the same to aclear extract, then stabilizing said extractby removing any remaining deleterious organisms.

13. The process which consists of propagating a fungus upon a culturemedium,

then lixiviating said mass withwater, and

concentrating the same to a clear extract,

then stabilizing said extract by removing any remaining deleteriousorganisms by. the .action of an antiseptic at suitable temperature.

14. As an article of manufacturean enzymic substance comprisingamaqueous extract from a mass of culture medium on which a fungus growthhas been' propagated and from which extract the decomposing-orfermenting bacteria have been removed, and containing an antiseptic. y

15."As a new article of manufacture an enzymic solution comprising anaqueous extract from a mass of culture medium on which a fungus growthhas been propa gated and containing sulfurous acid.

16.'As a new article of manufacture a stable enzymic solution is clearand transparent and free from decomposing or fermenting bacteria, ofslightly nutty taste and musty odor, possessing strongly diastatic andother enzymic properties, gives no coloration when an alcoholic solutionof gum guayacol and hydrogen peroxid is added thereto, and contains alarge amount of phosphate as a mineral constituent, a comparativelysmall amount of sugar and a fairly large amount of proteid organicmatter.

' 17. As 'a new article of manufacture, a stable enzymic solution freefrom decomposing or-fermenting bacteria, andwhich is clear andtransparent, of slightly nutty taste and mutty odor, possessing stronglydiastaticand other enzymic properties, gives stable, non-fermentingenzymic, amber colored solution, characterized by a somewhat nutty tasteand musty odor, and possessing strongly diastatic and otherenzymioproperfrom deleterious organisms and possessing ties. markeddiastatic and proteolytic properties.

19. As anew article of manufacture an l In testimony whereof we havehereunto 10 enzymic solution comprising an aqueous exset our hands onthis 22nd day of a'nuary,

5 tract from a mass of culture medium on A. D. 1918. which afungusgrowth has been propagated, JOKICHI TAKAMINE. said extract being high inphosphates, free J OKICHI TAKAMINE, JR.

